The document summarizes extraction methods for alkaloids from plants. It describes two main methods - solvent extraction under alkaline conditions, which extracts alkaloids as free bases using organic solvents, and extraction under acidic conditions, which extracts alkaloid salts into aqueous extracts that can then be made basic to recover the free bases. Both methods involve defatting the plant material first before extraction. The document provides details on the steps involved in each extraction method and notes that no universal quantification method exists for all alkaloids due to their chemical diversity.

1. Extraction, estimation and thin layer chromatography of alkaloids: A review
Gangwal A.
Smriti college of pharmaceutical education, Indore
Abstract
Since time immemorial alkaloids have been in use in various parts of the world for sure shot
treatment or for approximate and rudimentary treatment of various ailments. They were among
the earliest chemicals isolated in pure form from natural sources. Till date they are the most
fancied natural chemical for scientist of various back grounds (botanist, biochemist,
pharmacognosist , pharmacologist, natural product chemist etc), because they are one of the
most potent and promising bioactive chemicals, obtained from natural sources. Various
alkaloids have been reported in literature for various pharmacological activities like anti-cancer,
anti-cholinergic, hypotensive, analgesic to list a few. The quotable example is morphine which
has yet to find its rival for treatment of severe pain. Worldwide drug discovery is going through a
rough patch. Various models are being discussed, new approaches are emerging, synthetic
drugs are enjoying good position among available options. Still natural sources can never ne
undermined, as most of the mainline drug molecules trace their route to natural sources directly
or indirectly. In this review article an attempt has been made to compile basic information on
alkaloids from drug discovery point of view.
Introduction

2. Plant remains to be the enviable source of molecules of therapeutic significance. Since
antiquity, these bio resources have been in use for variety of diseases in different part of
the world. Regardless of the type of plant, targeted ailment or other such parameters,
the one step which is one of the most important and common is removal of the molecule
or fraction or part thereof from the plant biomass. Several new methods besides the
usual organic solvent extraction have been developed over the last few years for the
extraction of primary and secondary metabolites. These are alcohol extraction with
various biocompatible solvents, recovery of carboxylic acids and antibiotics with reactive
extraction, dissociation extraction, aqueous two-phase extraction, and supercritical and
near critical fluid extraction. Extraction and re-extraction processes are integrated into a
single step by emulsion liquid membrane and solid supported liquid membrane
extractions. (Extraction of primary and secondary metabolites.). There are several
extraction procedures or schemes (depending on various factors) for isolation of various
plant constituents generally known as primary and secondary metabolites, nonetheless
there are only one or two methods for scrupulous and perfect extraction of these
metabolites. Irrespective of the plant or part thereof or activity or subsequent operation,
these methods are sufficient to provide perfect extraction of various metabolites viz
alkaloids, flavonoids, tannins, saponins, carbohydrates etc. In various publications,
sometimes extraction schemes is not fully mentioned or not followed as mentioned in the
pioneering text source or there is reporting of some modified process. There is a need of
piled up information for the extraction, estimation and chromatography of some class of
phytoconstituents, especially for the researchers interested in exploring a plant afresh or
even for a routine assignment. This project is an attempt to compile and summarize the
most relevant and time tested procedures for three basic operations while studying a
plant from view point of phytochemistry or some allied reasons. To keep the text relevant
and limited, barring few instances direct methods are given. Extensively cited and used
procedures are being mentioned here. Many more procedures can be spotted in
literature. Variation might be in starting solvent or fractionation schemes but in most of
such cases ultimate steps usually remains same. This variation is intended because of
subsequent steps, chiefly isolation of pure phytochemicals from crude extract employing
range of solvents. Sometimes extraction is done to get rid of unwanted material for they
hinder the removal of other metabolite or they are to be separated later in the extraction
protocol or simply they are the problematic constituents in the sense they show false
positive chemical presence or false biological activities. Therefore this project also
describes the process to remove out rightly interfering compounds¹.
Extraction
Natural products may be obtained from the crushed biological material by extraction with a
solvent such as petroleum ether, chloroform (trichloroniethane), ethyl acetate (ethyl ethanoate)
or methanol. Several solvents of increasing polarity may be used. Thus lipid material (waxes,
fatty acids, sterols, carotenoids and simple terpenoids) can be extracted with non-polar solvents
such as petroleum ether, but more polar substances such as the alkaloids (mainly free bases)
and glycosides are extracted with methanol, aqueous methanol or even hot water. Many
alkaloids are present as their salts with naturally occurring acids such as tartaric acid. Polar
solvents dissolve ionic solutes and other polar substances. There are many methods based on
the technique or set up used but this project will explore only classical methods because such
methods are easy, putative and can be implemented in most of the laboratories in limited
setups. When it comes to extraction of phytoconstituents, the most widely employed method is
extraction using a single solvent at atmospheric pressure which can be boiled owing to their
azeotropic nature. Whether the compound(s) to be isolated is chemically undefined or not, it is
important to have an idea about the relationship between the method applied and the properties
of the substance extracted. A well known and time tested thumb rule is that “like dissolves like”.

3. It means non polar solvents will remove non polar phytoconstituents and vice versa holds
equally true. In most instances it is likely that that moderately polar phytoconstituents will be
extracted ²,
³. Extractions can be either ‘‘selective’’ The initial choice of the most appropriate
solvent is based on its selectivity for the substances to be extracted. In a selective extraction,
Thus non polar solvents are used to solubilize mostly Lipophilic compounds (e.g., alkanes, fatty
acids, pigments, waxes, sterols, some terpenoids, alkaloids, and coumarins). Medium-polarity
solvents are used to extract compounds of intermediate polarity (e.g., some alkaloids,
flavonoids), while more polar ones are used for polar compounds (e.g., flavonoid glycosides,
tannins, some alkaloids). Water is not used often as an initial extractant, even if the aim is to
extract water-soluble plant constituents (e.g., glycosides, quaternary alkaloids, tannins) ¹‚
²‚
³. A
crude natural product extract is generally an extremely complicated mixture of several
compounds possessing varying chemical and physical properties. The fundamental strategy for
separating these compounds is based on their physical and chemical properties that can be
cleverly exploited to initially separate them into various chemical groups. However, in some
cases, from the literature search of the related genera and families, it is possible to predict the
types of compounds that might be present in a particular extract. This tentative prediction on the
possible identity of the classes of compounds may help choose suitable extraction and
partitioning methods, and solvents for extracting specific classes of compounds, for example,
phenolics, saponins, alkaloids. Plant natural products are usually extracted with solvents of
increasing polarity, for example, first n-hexane, diethylether, chloroform (CHCl₃), to name a few,
followed by more polar solvents, i.e., methanol (MeOH), depending on the chemical and
physical nature of the target compounds. Alcoholic (MeOH or EtOH) extracts of plant materials
contain a wide variety of polar and moderately polar compounds. By virtue of the co-solubility,
many compounds, which are insoluble individually in pure state in MeOH or EtOH, can be
extracted quite easily with these solvents. The concentrated extract is then extracted with an
equal volume of n-hexane, usually three times, to give a fraction containing non-polar
compounds, such as lipids, chlorophylls, and so on. The process is sometimes referred to as
‘‘defatting.’’ Although MeOH and n-hexane are not completely miscible, they are miscible to
some extent. Sometimes, a small amount of water is added to MeOH to obtain a 95%-aqueous
methanolic solution to get two distinct layers with similar volumes. The methanolic layer is
evaporated to dryness and then dissolved in water. Occasionally it is not a solution, but a
suspension. The solution (suspension) is partitioned between CHCl₃, ethylacetate (EtOAc), and
n-butanol (n-BuOH), successively. Partitioning with CHCl₃ can be omitted depending on the
chemical nature of the target compounds. Less polar compounds are present in the CHCl₃
soluble fraction and polar compounds, probably up to monoglycosides, in the EtOAc-soluble
one. The n-BuOH fraction contains polar compounds, mainly glycosides. Evaporation of the
remaining water layer leaves polar glycosides and sugars as a viscous gum. However,
separation by solvent partitioning cannot be always performed in a clear cut manner;
overlapping of the compounds in successive fractions is usually found. When using EtOAc as
an extraction solvent, especially the technical grade solvent, researchers must remember that it
contains a trace amount of acetic acid (AcOH), which may cause a trans-esterification of acetyl
group to the hydroxyl groups, and have a catalytic effect on labile functional groups or delicate
structures. When the acetates of some compounds are isolated from the EtOAc-soluble or
subsequent n-BuOH-soluble fraction, it is suspected that trans-esterification may have produced
the acetates of the original compounds as artifacts. Chloroform is an ideal solvent for extracting
alkaloids owing to its slight acidic nature, because alkaloids tend to be soluble in acidic media.
When water layer is to be extracted thoroughly with n-BuOH, n-BuOH saturated with water is
frequently used. Although n-BuOH is not miscible with water, 9.1ml of n-BuOH is soluble in
100ml of water at 250
C. Therefore, when the water layer is extracted with n-BuOH unsaturated

4. with water many times, the volume of the water layer drastically decreases. Usage of
unbalanced volumes of solvents sometimes causes unexpected partitioning of compounds.
When saponins are the major target, it is advisable that the glycoside fraction (n-BuOH layer) is
partitioned with a 1%-KOH solution to remove widely distributed phenolic compounds, such as
flavonoids and related glycosides. Before concentrating the extract, the n-BuOH layer must be
washed several times with water. In turn, re-extraction of the acidified alkaline layer gives a
fraction rich in phenolic compounds. Some acylated saponins and flavonoids, present in plant
extracts, are also hydrolyzed under alkaline conditions. Thus, at least a small-scale pilot
experiment, such as tracing the fate of compounds by thin layer chromatography (TLC), is
strongly recommended. However, this method is useful for the isolation of known alkali-resistant
saponins on a large scale. Partitioning between Miscible Solvents Contrary to what has already
been discussed earlier, miscible solvents are sometimes used for partitioning on addition of
water. A plant material is extracted with MeOH and evaporated to obtain a residue. The residue
is re-dissolved in 90% aqueous MeOH, and the resulting solution is extracted with n-hexane.
This step seems to be similar to the previous partitioning example. In the next step, an
appropriate amount of water is added to the 90%-aqueous MeOH to obtain an 80% aqueous
solution, which is then extracted with CCl₄ (MeOH and CCl₄ are miscible). The final step is to
make a 65%-aqueous MeOH solution with the addition of water, and the resulting solution is
extracted with CHCl₃ (MeOH and CHCl₃ are miscible). Evaporation of the n-hexane, CCl₄, and
CHCl₃ layers gives three fractions in order of polarity. Concentration of the 65%-aqueous MeOH
layer gives the most polar fraction. This fraction is expected to contain glycosides as major
constituents as well as a large amount of water-soluble sugars. preparation of detannnified
extract: Defatted methanolic extract is partitioned with chloroform. The chloroform extract is
washed with 1% NaCl to get extract tannin. Some authors have suggested the removal of crude
saponins, from n butanol fraction of defatted menthol or alcohol or hydro-alcoholic extract, by
precipitating with ethyl acetate².
ALKALOIDS
Extraction
Being bases, alkaloids are normally extracted from plants into a weakly acid (1M HCl or 10%
acetic acid) alcoholic solvent and are then precipitated with concentrated ammonia. These steps
may be repeated or further purification can be achieved by solvent extraction. Such relatively
crude extracts can be tested for the presence of alkaloids by applying various reagents meant
for these secondary metabolites which represent the largest single class of secondary plant
substances. The extraction of alkaloids is based, as a general rule, on the fact that they
normally occur in the plant as salts and on their basicity, in other words on the differential
solubility of the bases and salts in water and organic solvents. The plant material often contains
substantial quantities of fats, and also waxes, terpenes, pigments, and other lipophilic
substances which may interfere with extraction procedure, for example, by causing the
formation of emulsion. These technical problems can be more or less completely avoided by a
preliminary defatting of the powdered drug. Petroleum ether and hexane are well suited for this
step: alkaloids are soluble in these solvents only in exceptional cases, when medium is neutral.
The methods for the isolation of alkaloids are based on the fact that they can be extracted under
neutral or basic conditions (after basification of plant material to pH 7-9 with ammonia, sodium
carbonate, or sodium bicarbonate), as free base with organic solvents (e.g., dichloromethane,
chloroform, ethers, ethyl acetate, alcohols) and as protonated base with polar solvent (water,
alcohols) under acidic conditions (after acidification to pH 2-4 with diluted acids like phosphoric
acid, sulphuric acid, citric acid)³. Keeping all these things constant, two processes are used
routinely for the removal of alkaloids from plants.

5. A) Solvent extraction in alkaline medium The powdered material is moistened with water and
mixed with lime which combines with acids, tannins and other phenolic substances and sets
free the alkaloids (if they exist in the plant as salts). Extraction is then carried out with organic
solvents such as its ether or petroleum spirit to take free bases. The concentrated organic liquid
is then shaken with aqueous acid and allowed to separate. Alkaloid salts are now in the
aqueous liquid, while many impurities (usually neutral) remain behind in the organic liquid. The
operation is repeated as many times as necessary until the organic phase no longer contains
any alkaloids, which can e confirmed by applying various qualitative chemical tests meant for
alkaloids. The aqueous solutions of the alkaloid salts, combined, and washed with a non polar
solvent (hexane, diethyl ether). These are alkalinized with a base in the presense of an organic
solvent and not miscible the alkaloids as bases precipitate and dissolve in the organic phase.
The extraction of the aqueous phase continues until the totality of the alkaloids has gone into
the organic phase. The purification step may be carried out, like the previous one and
depending on the quantity. Finally, the organic solvent containing the alkaloids as bases is
decanted, freed from possible traces of water by suitable drying agent and evaporated under
reduced pressure. A dry residue is left.
B) Extraction in acidic medium: The powdered material is extracted with water or aqueous
alcohol containing dilute acid. Pigments and other unwanted materials are removed by shaking
with chloroform or other organic solvents. The free alkaloids are then precipitated by the
addition of excess sodium bicarbonate or ammonia and separated by filtration or by extraction
with organic solvents. This technique can be used to extract quaternary ammonium salts. The
alkaline medium ensures that the alkaloids are in their free base form. Medium polarity
alkaloidal bases can be extracted using such organic solvents as chloroform, dichloromethane
or diethyl ether. In this method alkaloidal salts are formed, which are ionized and therefore
soluble in aqueous media. The alkaloid then can be recovered as free base by making the
environment basic of aqueous extract²‚
⁶‚
⁷.
Estimation of alkaloids
There is no universal method which can be applied to quantify analytically all the classes of
alkaloids. Thus alkaloids in the free base form are difficult to crystallize whereas their salts
crystallize comparatively easily. A rapid, easy, and simple spectrophotometric method was
developed for the estimation of total alkaloids precipitated by Dragendorff's reagent in plant
materials. It is based on the formation of yellow bismuth complex in nitric acid medium with
thiourea. The yellow-colored complex formed obeys Lambert-Beer's law in the concentration
range of 0.06-50 micro g/ml with λ at 435 nm. Using this method, the alkaloidal percentage of
certain alkaloids (ajamalicine, papaverine, cinchonine, piperine, berberine) and some plant
materials containing alkaloids (Berberis aristata, Solanum nigrum, and Piper longum³‚
⁸‚
⁹.
Thin Layer Chromatography (Silica gel 60 F 254 pre-coated TLC plates)
Toluene-ethyl acetate-diethylamine (70:20:10) is suitable for most of the drugs. Most of the
alkaloids are separated on silicic acid. Aluminium oxide-precoated TLC plates may also be
employed. With Dragendorff reagent alkaloids spontaneously give orange-brown color, which is
usually stable in visible light. Dragendorff reagent followed by spay with sodium nitrite can also
be tried to intensify the color developed by former reagent. Extract dried tissue from the plant
with 10% acetic acid in ethanol, leave to stand for few hours. Concentrate the extract to one-
quarter of the original volume and alkaloids are then precipitated by drop wise addition of
concentrated NH4OH. Former is collected by centrifugation, washing with 1%NH4OH. Residue is
then dissolved in ethanol or chloroform. Commonly employed and most informative solvent
systems and other requirement are as under: Methanol: Con. NH4OH (200:3) and n-butanol-
aqueous citric acid (on sodium citrate-buffeted paper) Detection of the spots: Presence of

6. alkaloids is ascertained by any fluorescence in UV light and then by application of following
spray reagents separately: Dragendorff, iodoplatinate and Marquis ⁵,
¹¹.
CONCLUSION
Plant secondary metabolites are currently the subject of much research interest, but their
extraction as part of phytochemical or biological investigations presents specific challenges that
must be addressed throughout the solvent extraction process. Successful extraction begins with
careful selection and preparation of plant samples, and thorough review of the appropriate
literature for indications of which protocols are suitable for a particular class of compounds or
plant species. During the extraction of plant material, it is important to minimize interference
from compounds that may coextract with the target compounds, and to avoid contamination of
the extract, as well as to prevent decomposition of important metabolites or artifact formation as
a result of extraction conditions or solvent impurities. This chapter presents an overview of the
process of plant extraction, with an emphasis on common problems encountered and methods
for reducing or eliminating these problems. In addition to generally applicable extraction
protocols, methods are suggested for more or less selectively extracting specific classes of
compounds, and phytochemical methods are presented for detection of classes of compounds
commonly encountered during plant extraction, including selected groups of secondary
metabolites and interfering compounds. Plants and microorganisms produce complex mixtures
of natural products, and the election of the best protocol for an efficient extraction of these
substances is not a simple task. ‘‘Classic’’ solvent-based procedures (e.g., maceration,
percolation, Soxhlet extraction, extraction under reflux, steam distillation) are still applied widely
in phytochemistry despite the fact that they lack reproducibility and are both time- and
solventconsuming. This is principally because they only require basic glassware and are
convenient to use for both initial and bulk extraction. Accelerated solvent extraction is a newer
instrumental technique. While it offers some advantages over conventional methods (mainly
efficiency and reproducibility), it is best suited for initial rather than bulk extraction. It has found a
wider application in industry (where large numbers of extracts have to be produced in an
efficient and reproducible way) rather than in academia. To date, mainly plant and microbial
sources have been investigated for their metabolites. However, it is important to remember that
researchers are only beginning to explore other biotopes (e.g., the marine environment, insects)
and that many plants and microorganisms have not yet been characterized. Moreover, several
species among the bacteria known are yet to be cultured under laboratory conditions. This
leaves much scope for the potential discovery of novel and/or useful natural products in the
future¹.
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